Transcriptome analysis of a respiratory Saccharomyces cerevisiae strain suggests the expression of its phenotype is glucose insensitive and predominantly controlled by Hap4, Cat8 and Mig1

Abstract

Background: We previously described the first respiratory Saccharomyces cerevisiae strain, KOY.TM6*P, by integrating the gene encoding a chimeric hexose transporter, Tm6*, into the genome of an hxt null yeast. Subsequently we transferred this respiratory phenotype in the presence of up to 50 g/L glucose to a yeast strain, V5 hxt1-7Delta, in which only HXT1-7 had been deleted. In this study, we compared the transcriptome of the resultant strain, V5.TM6*P, with that of its wild-type parent, V5, at different glucose concentrations.

Results: cDNA array analyses revealed that alterations in gene expression that occur when transitioning from a respiro-fermentative (V5) to a respiratory (V5.TM6*P) strain, are very similar to those in cells undergoing a diauxic shift. We also undertook an analysis of transcription factor binding sites in our dataset by examining previously-published biological data for Hap4 (in complex with Hap2, 3, 5), Cat8 and Mig1, and used this in combination with verified binding consensus sequences to identify genes likely to be regulated by one or more of these. Of the induced genes in our dataset, 77% had binding sites for the Hap complex, with 72% having at least two. In addition, 13% were found to have a binding site for Cat8 and 21% had a binding site for Mig1. Unexpectedly, both the up- and down-regulation of many of the genes in our dataset had a clear glucose dependence in the parent V5 strain that was not present in V5.TM6*P. This indicates that the relief of glucose repression is already operable at much higher glucose concentrations than is widely accepted and suggests that glucose sensing might occur inside the cell.

Conclusion: Our dataset gives a remarkably complete view of the involvement of genes in the TCA cycle, glyoxylate cycle and respiratory chain in the expression of the phenotype of V5.TM6*P. Furthermore, 88% of the transcriptional response of the induced genes in our dataset can be related to the potential activities of just three proteins: Hap4, Cat8 and Mig1. Overall, our data support genetic remodelling in V5.TM6*P consistent with a respiratory metabolism which is insensitive to external glucose concentrations.

Figure 1

An overview of our dataset in the context of key yeast metabolic pathways. Genes with altered expression in V5.TM6*P compared with V5 are marked as follows: induced genes are denoted in large, bold font; down-regulated genes are crossed out. Genes that were previously found to be altered in studies of respiratory-state yeast [6], but which are not found in our dataset are denoted in small, grey text. The chimeric Tm6*p transporter, which comprises the amino-terminal half of Hxt1 and the carboxy-terminal half of Hxt7, is responsible for the respiratory phenotype of V5.TM6*P at high glucose concentrations and is present in the plasma membrane. The grey boundaries represent the plasma membrane (top) and the mitochondrial membrane (left). ^†Note that ICL(1.5), NDE1 (1.8) and GPD1 (1.8) are induced in our study (see Additional file 3). ^‡ LPD1 is a component of pyruvate dehydrogenase.

Figure 2

An overview of our dataset in the context of the respiratory chain in yeast. Genes with altered expression in V5.TM6*P compared with V5 are marked as follows: genes induced by a factor ≥2.0 are denoted in large, bold font; genes induced by a factor <2.0 are denoted in smaller, grey font.

Figure 3

(A) The influence of multiple Hap complex sites on the change in gene expression of the genes in Additional file 1. A Fisher's exact test was performed for genes containing predicted binding sites for the Hap complex and their association with induction or repression. Genes were grouped into either those with one Hap complex binding site or those with two or more according to Additional file 1. (B) The influence of Hap4, Cat8 and Mig1 on the magnitude of induction of the genes in Additional file 1. The average fold-induction of genes in Additional file 1, grouped according to the transcription factor binding sites they contain, was calculated. It was observed that those containing binding sites for each of the Hap complex (where Hap4 is the activator), Cat8 and Mig1, had the highest average fold induction with Mig1 being a dominant factor in high induction. A bar for Cat8 is not included as only one gene (YOR019W, factor change 2.1) has a Cat8 site alone. (C) A Venn diagram of the distribution of binding sites for the Hap complex, Cat8 and Mig1 in the genes in Additional file 1. 88% of the genes in Additional file 1 contain a binding site for one or more of the Hap complex, Cat8 and Mig1. The range of induction for the 190 genes from Additional file 1 is 2.0 to 21.7.

Figure 4

Transcript dependence on external glucose concentration for 20 genes in the V5 strain. A clear glucose-dependence (grey bars) was observed for all genes in the V5 strain with only a small variation in array signal (white bars) for the same genes in V5.TM6*P. Genes marked with a dot also have a significant change of expression on going from V5 to V5.TM6*P and as such are listed in Additional file 1. The factor change in gene expression was obtained as the quotient between the expression values at 37 and 9 g/L glucose given by the line fitted to the expression data for each gene. R² values are for the linear fit for the V5 strain. The R² values for V5.TM6*P were between 0.16 and 0.78 indicating no significant linear relationship.